Flavonoid compositions and related methods

ABSTRACT

Flavonoids that are isolated from plant material of the genus Cecropia can be used to perturb G-protein coupled receptors in a mammalian cell. In some instances, one or more flavonoids may interact with one or more of the G-protein coupled receptors to transiently increase the concentration of cytosolic calcium. Administration of the isolated flavonoids can be used to treat hypertension, to protect the integrity of blood vessels and related conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/884,118, filed Jan. 30, 2018, and entitled FLAVONOIDCOMPOSITIONS AND RELATED METHODS, which claims the benefit of U.S.Provisional Application No. 62/453,928, filed Feb. 2, 2017, both ofwhich hereby incorporated by reference.

TECHNICAL FIELD

The present disclosure relates generally to the field of bioactivecompounds. More particularly, some embodiments relate to compositions,formulations, and methods that involve compounds that can be extractedfrom plants of the genus Cecropia, such as flavonoid-containingcompositions.

BRIEF DESCRIPTION OF THE DRAWINGS

The written disclosure herein describes illustrative embodiments thatare non-limiting and non-exhaustive. Reference is made to certain ofsuch illustrative embodiments that are depicted in the figures, inwhich:

FIG. 1 is an ESI/MS spectrum (positive ion mode) of compound P9 (i.e.,isovitexin-2″-O-rhamnoside).

FIG. 2 is an ESI/MS/MS spectrum (positive ion mode) of fragment ionm/z=601 of compound P9.

FIG. 3 is an ESI/MS/MS spectrum (positive ion mode) of fragment ionm/z=579 of compound P9.

FIG. 4 is an ESI/MS spectrum (positive ion mode) of compound P7 (i.e.,isovitexin-2″-O-glucoside).

FIG. 5 is an ESI/MS/MS spectrum (positive ion mode) of fragment ionm/z=617 of compound P7.

FIG. 6 is an ESI/MS/MS spectrum (positive ion mode) for fragment ionm/z=595 of compound P7.

FIG. 7 is an ESI/MS spectrum (positive ion mode) of compound P8 (i.e.,isovitexin-2″-O-xyloside).

FIG. 8 is an ESI/MS/MS spectrum (positive ion mode) of fragment ionm/z=587 of compound P8.

FIG. 9 is an ESI/MS/MS spectrum (positive ion mode) of fragment ionm/z=565 of compound P8.

FIG. 10 is an ESI/MS spectrum (positive ion mode) of compound P6 (i.e.,isovitexin-O-xyloside).

FIG. 11 is an ESI/MS/MS spectrum (positive ion mode) of fragment ionm/z=587 of compound P6.

FIG. 12 is an ESI/MS/MS spectrum (positive ion mode) of fragment ionm/z=565 of compound P6.

FIG. 13 is a ¹H NMR spectrum of compound P6.

FIG. 14 is a MALDI/TOF spectrum (positive ion mode) of compound P4(i.e., isoorientin-2″-O-xyloside).

FIG. 15 is a MALDI/MS/MS spectrum (positive ion mode) of fragment ionm/z=581 of compound P4.

FIG. 16 is a MALDI/TOF/TOF spectrum (positive ion mode) of fragment ionm/z=329 of compound P4.

FIG. 17 ESI/MS spectrum (positive ion mode) of compound P2 (i.e.,chlorogenic acid)

FIG. 18 is an UV chromatogram of chlorogenic acid and compound P2.

FIG. 19 is a bar graph that shows relative intracellular calciumconcentrations of CHO-AEQ-AT₂ cells that have been exposed to variouscompositions.

FIG. 20 is a bar graph that shows relative intracellular calciumconcentrations of CHO-AEQ-ET_(B) cells that have been exposed to variouscompositions.

FIG. 21 is a bar graph that shows relative intracellular calciumconcentrations of CHO-AEQ-AT₁ cells that have been exposed to variouscompositions.

BACKGROUND

Trees in the genus Cecropia (in the Urticaceae family) are generallydioecious, with few branches (usually with a candelabrum-like branchingsystem), and a hollow trunk. These trees can have stilt roots, fullyamplexicaul stipules, peltate blades with one to two trichilia at thebase of the petioles, and inflorescences arranged in digitate clusters(or a single inflorescence), usually enveloped by a spathe untilanthesis. In such trees, the interfloral bracts are generally absent,the flowers have two stamens, and the trees have small, dry fruitsenveloped by a tubular greenish perianth (Berg et al., Flora 51 Neotrop1-208 (1990); Berg & Roselli 94 Flora Neotrop. 1-230 (2005)). Treeswithin this genus are widespread and abundant. For instance, thesegenerally fast-growing trees are distributed across the tropical andsubtropical rainforests of Mexico, Central America, and South America atelevations below 2600 m (Franco-Rosselli & Berg, 19 Caldasia 285-296(1997). The genus includes 61 species (Berg & Roselli 94 Flora Neotrop.1-230 (2005), including species popularly known, among other folk names,as “yarumo,” “guarumo,” “guarumbo,” “embaúba,” “ambay,” “torém,” and“trumpet tree.” (Luengas-Caicedo et al., 62 J Biosci. 701-709 (2007);Costa et al., 22 J Braz Chem Soc. 1096-1102 (2011); Ospina Chávez J, etal., 42 L. Rev Colomb Cienc Quim Farm. 244-259 (2013); Hernández et al.,18 Rev Cuba Plantas Med. 586-595 (2013); Montoya Peláez et al., 23Brazilian J Pharmacogn. 754-761 (2013).)

Trees within the genus Cecropia are of ecological significance. Forinstance, due to their rapid rate of growth, such trees are often theprimary colonizers of deforested tropical areas (Monro et al., availableathttp://www.kew.org/science/tropamerica/neotropikey/families/Urticacea.htm(2009)) and act as invasive species in non-native regions. (Conn et al.,57 Blumea J Plant Taxon Plant Geogr. 136-142 (2012); GISD, available athttp://www.iucngisd.org/gisd/species.php?sc=116.) In addition, mostspecies within the genus Cecropia are ant-plants or myrmecophytes. Inother words, such trees may live in a mutualistic relationship with acolony of symbiotic ants, especially ants of the genus Azteca. Theypossess specialized structures for offering shelter and food to ants inexchange for protection against natural enemies. (Dejean et al., 97Naturwissenschaften. 925-934 (2010); Oliveira et al., 10 PLoS One 1-13(2015).)

Medicinal claims related to the genus Cecropia have been advanced inseveral Latin American countries. Material from such plants has beenused as a diuretic, an antioxidant, an antitussive, an expectorant, andfor the treatment of several ailments or diseases such as cough, asthma,diabetes, inflammation, anxiety, and depression. (Costa et al., 22 JBraz Chem Soc. 1096-1102 (2011); Gazal et al., 108 Brain Res Bull. 10-17(2014); Pacheco et al., Biomed Res Int. 1-10 (2014). Reports have alsobeen made purporting the efficacy of plant-derived material in woundhealing, pain relief, and antimicrobial activity. Souccar et al., 15Phytomedicine 462-469 (2008). The therapeutic properties of these plantshave generally been attributed to compounds in the plants, such asflavonoids, proanthocyanidins (Luengas-Caicedo et al., 62 J Biosci.701-709 (2007)), terpenoids, steroids (Ospina Chávez J, et al., 42 L.Rev Colomb Cienc Quim Farm. 244-259 (2013)), chlorogenic- and caffeicacid (Davet et al., 185 J Ethnopharmacol 255-262 (2016)), and otherphenolic compounds (Gazal et al., 108 Brain Res Bull. 10-17 (2014)).

Flavonoids are a large group of secondary metabolites. The carbonstructure of such compounds can be abbreviated C6-C3-C6, with twoaromatic rings and a cycled oxygen. They can be found as aglycones,glycosides, and methyl derivatives at different positions of their corestructure. The most abundant flavonoid glycosides are the flavones O/Cglycosides and the flavonol O-glycosides. The glycosidic bond isnormally found at position 3 or position 7, and the carbohydrate unitcan be a glucoside, galactoside, ramnoside, or arabinoside. Thebiological activities of these compounds can depend not only on thestructural differences of the core but also on their glycosylationpatterns.

DETAILED DESCRIPTION

This disclosure is related to bioactive compositions, formulations, andmethods that involve compounds that can be extracted from plants of thegenus Cecropia, such as flavonoid compositions.

It will be readily understood that the embodiments, as generallydescribed herein, are exemplary. The following more detailed descriptionof various embodiments is not intended to limit the scope of the presentdisclosure, but is merely representative of various embodiments.Moreover, the order of the steps or actions of the methods disclosedherein may be changed by those skilled in the art without departing fromthe scope of the present disclosure. In other words, unless a specificorder of steps or actions is required for proper operation of theembodiment, the order of specific steps or actions may be modified.

Amounts, concentrations, and other numerical data may be presentedherein in a range format. It is to be understood that such range formatis used merely for convenience and brevity and should be interpretedflexibly to include not only the numerical values explicitly recited asthe limits of the range, but also all the individual numerical values orsub-ranges encompassed within that range, as if each numerical value andsub-range were explicitly recited. For example, an amount of from 1 mgto 200 mg should be interpreted to include not only the explicitlyrecited limits of 1 mg and 200 mg, but also individual amounts such as 2mg, 3 mg, 4 mg, and sub-ranges such as 10 mg to 50 mg, 20 mg to 100 mg,etc. Unless otherwise stated, all ranges include both endpoints.

Some embodiments of the present disclosure relate to methods ofperturbing (e.g., acting as an agonist or antagonist for) one or moreG-protein coupled receptors in a mammalian cell. In some embodiments,such methods involve obtaining and/or producing a formulation thatcomprises one or more flavonoids selected from the group consisting ofisovitexin-2″-O-rhamnoside, isovitexin-2″-O-glucoside,isovitexin-2″-O-xyloside, isovitexin-O-xyloside, andisoorientin-2″-O-xyloside. Such flavonoid(s) may be derived from plantmaterial of the genus Cecropia.

For example, in some embodiments, the formulation is obtained byextracting the one or more flavonoids from plant material of the genusCecropia. In some embodiments, the flavonoids from the plant materialare isolated from exclusively (or in greater abundance) from pistillateflowers (female) plants of the genus Cecropia. The extraction may becarried out via any suitable solvent. For example, in some embodiment,the solvent used to carry out the extraction may be selected from thegroup consisting of water, alcohols, ketones, esters, ethers, polyhydricalcohols, chlorine-containing solvents, and mixtures of at least two ofthe aforementioned solvents. Stated differently, in some embodimentsplant material from the Cecropia genus may be mixed with one or moresolvents to extract a portion of the plant material into the one or moresolvents. In some embodiments, the solvent or solvent system is anorganic solvent. In some embodiments, the solvent comprises or consistsof an alcohol, such as methanol, ethanol, or butanol (e.g., alcoholswith four or fewer carbon atoms). The extracted portion of the plantmaterial can be isolated from the remainder of the plant material.Subsequently, at least a portion of the solvent(s) may be removed toobtain a first composition. In some embodiments, the extractedcomponents are further purified (e.g., via chromatography or afractionated liquid/liquid or liquid/solid extraction technique).

In some embodiments, the plant material from the genus Cecropia may bemacerated prior to extracting the one or more flavonoids from the plantmaterial. In some embodiments, the plant material is exclusively fromaerial parts of the plant (e.g., the leaves and stems) and does notinclude roots. In some embodiments, the plant material is from one ormore plants of the species Cecropia obtusifolia.

In some embodiments, the first composition obtained by extraction may becombined with a second composition to form a formulation foradministration to mammalian cells and/or a mammalian subject. In someembodiments, the first composition is between 0.001% and 100% of theformulation by weight, between 0.001% and 20% of the formulation byweight, and/or between 0.1% and 3% of the formulation by weight.

In some embodiments, the one or more flavonoids are between 0.1% and50%, such as between 0.3% and 15% of the formulation by weight. In someembodiments, the formulation includes two or more, three or more, fouror more, or all five of the flavonoids selected from the groupconsisting of isovitexin-2″-O-rhamnoside, isovitexin-2″-O-glucoside,isovitexin-2″-O-xyloside, isovitexin-O-xyloside, andisoorientin-2″-O-xyloside. In some embodiments, the flavonoids of theformulation include or consist of isovitexin-2″-O-xyloside andisovitexin-2″-O-rhamnoside. In some embodiments, the formulationconsists essentially of the one or more flavonoids. In other words, theformulation may include one or more flavonoids that are configured toperturb one or more G-protein coupled receptors, but does not includeother compounds or substances that materially affect the ability of theformulation to perturb the G-protein coupled receptors. In someembodiments, the formulation consists essentially ofisovitexin-2″-O-xyloside and isovitexin-2″-O-rhamnoside. In someembodiments, the formulation includes one or more of a dispersant, ahumectant, a carrier, an antistatic agent, a filler, or a diluent. Insome embodiments, the formulation includes a total Cecropia extract,which includes all active extractable components of the plant. In otherembodiments, the extract is further purified to obtain a fraction thatincludes crude, semi-purified, or purified flavonoids. In someembodiments, only the active components of the extract are used in theformulation.

The formulation may be delivered to one or more mammalian cells toperturb one or more G-protein coupled receptors. In other words, themammalian cells may be contacted with an effective amount of theformulation. For example, in some embodiments, a cell may be identifiedin which perturbation of one or more G-protein coupled receptors isdesired. For example, in some embodiments, the formulation is deliveredto cells in vitro. In other embodiments, the formulation is delivered inan effective amount to a mammalian (e.g., human) patient, such as apatient in which perturbation of G-protein coupled receptor is desired.In some embodiments, such patients may have been selected for treatmentbased on a diagnosis of high blood pressure or an elevated risk of highblood pressure.

The one or more G-protein coupled receptors that are perturbed by theformulation may be selected from the group consisting of (1) angiotensinII receptor, type 1, (2) angiotensin II receptor, type 2, and (3)endothelin receptor type B. In some embodiments, the one or moreflavonoids of the formulation are, individually or collectively,configured to perturb all three of (1) the angiotensin II receptor, type1, (2) the angiotensin II receptor, type 2, and (3) the endothelinreceptor type B. In some embodiments, the formulation acts as an agoniston the AT₂ and ET_(B) receptors, and antagonizes the AT₁ receptor. Insome embodiments, perturbation of the one or more G-protein coupledreceptors causes vasodilation in a mammalian subject. Stateddifferently, administration of the formulation to a patient may cause adecrease in blood pressure. In other or further embodiments,administration to a patient may provide neuroprotective,cardioprotective, and/or vasculoprotective effects. In some embodimentsthat include two or more flavonoids, the flavonoids may provide asynergistic effect on the one or more G-protein coupled receptorsrelative to the same quantity of each of the two or more flavonoidsalone.

Examples

Extraction of Plant Material and Isolation and Characterization ofActive Compounds

Leaves and stems of dried Cecropia obtusifolia were ground to obtain122.13 g of pulverized plant material. The pulverized plant material wasthen exhaustively extracted with three liters of methanol. The resultingalcohol extract was washed with 750 ml of hexane and two liters ofdichloromethane under agitation. The solvent was then removed underreduced pressure at temperatures of less than 40° C. to afford a drysolid.

The dry alcohol fraction from C. Obtusifolia (1 g) was submitted to acolumn chromatography over Sephadex LH-20 (45 cm×1.5 cm i.d, 10 g),using a mobile phase of water:ethanol (1:1). Ten fractions werecollected. Fraction 3 and fraction 4 were pooled (“Fraction 3-4”) basedon their HPLC profile, yielding 308.8 mg of solid material.

The chemical compounds of the solid material from Fraction 3-4 were thenisolated by semi-preparative reverse phase liquid chromatography (HPLC)to yield five pure compounds. More particularly, to isolate the chemicalcompounds of the solid material from Fraction 3-4, the sample wasapplied using a manual injector and separated on a C18 column (250mm×10.0 mm i.d.; 5 μm) at 40° C. The mobile phase was a gradientgenerated by combining solvent A (1% acetic acid, adjusted to pH 3.0)and solvent B (acetonitrile) as follows: 0-30 min, linear gradient fromA:B (90:10 v/v) to A:B (85:15 v/v); 30-45 min isocratic A:B (85:15 v/v).The flow rate was 2.0 mL/min. For detection, a chromatogram was recordedat 340 nm using a diode array detector (DAD) while the UV spectrum wasmonitored over a range of 200-500 nm.

The five compounds that were separated by HPLC were identified based ontheir mass spectrometry data, nuclear magnetic resonance (NMR) spectrums(¹H NMR, ¹³C NMR), and by comparison with a reference standard and/orthe available literature data. The data used for identification of thesecompounds is set forth below.

Compound P9

As shown in both FIG. 1 and Table 1, when subjected to ESI/MS (positiveion mode), compound P9 displayed a molecular ion (MI) at m/z 579.3[M+H]⁺ and a base peak (BP) at m/z 601.4 [M+Na]⁺. Its molecular formulawas deduced as C₂₇H₃₀O₁₄. Fragmentations of the molecular ion (FIG. 3)and the base peak (i.e., FIG. 2) produced abundant ions Yo⁺; 433.0[(M+H)-146]⁺ and 455.3 [(M+Na)-146]⁺. Such masses are attributed to lossof a neutral sugar moiety (deoxyhexose) from glycosylated hydroxylgroups. Ferreres et al., 1161 J Chromatogr A 214-23 (2007); Waridel etal., 926 J Chromatogr A. 29-41 (2001). Ions typical of C-hexosylflavones (see FIGS. 2-3 and Table 1) were also observed: ^(0.3)X₀ ⁺:365.3 [(M+Na)-146-90]⁺, and ^(0.2)X₀ ⁺: 335.3 [(M+Na)-146-120]⁺, and313.1 [(M+H)-146-120]⁺ (Ferreres et al., 1161 J Chromatogr A 214-23(2007); Waridel et al., 926 J Chromatogr A. 29-41 (2001)).

TABLE 1 Experimental data No. ESI(+)-MS ESI(+)-MS/MS  1 601.4 [M + Na]⁺601.3 [M + Na]⁺  2 583.4 [(M + Na)-H₂0]⁺  3 481.3 [(M + Na)-120]⁺  4455.3 [(M + Na)-146 (Rha)]⁺→ Y_(o) ⁺  5 437.3 [(M + Na)-Rha-H₂0]⁺  6365.3 [(M + Na)-Rha-90]⁺→ ^(0,3)X₀ ⁺  7 335.3 [(M +Na)-Rha-120]⁺→^(0,2)X₀ ⁺  8 579.3 [M + H]⁺ 579.3 [M + H]⁺  9 433.0 [(M +H)-146 (Rha)]⁺→ Y_(o) ⁺ 10 415.1[(M + H)-Rha-H₂O]⁺ 11 367.0[(M +H)-Glu-66]⁺→^(2,3)X⁺-2H₂O 12 313.1[(M + H)-Rha-120]⁺→^(0,2)X₀ ⁺

Compound P9 was also characterized by both ¹H NMR and ¹³C NMR. Theexperimental results are shown in Table 2 alongside published data forisovitexin-2″-O-rhamnoside (Prinz et al., 4 Chem Biodiverse. 2920-31(2007)).

TABLE 2 Published data Isovitexin-2″-O- rhamnoside (Prinz et al., 4 ChemBiodiverse. Experimental data 2920-31 (2007)) ¹H δ (ppm) ¹³C δ (ppm) ¹Hδ (ppm) ¹³C δ (ppm) DMSO-d₆, DMSO-d₆, DMSO-d₆, DMSO-d₆, No. 400 MHz 100MHz 400 MHz 100.6 MHz  2 163.32 163.5  3 6.73 (1H, s) 102.55 6.76 (s)102.9  4 181.99 181.9  5 13.53 159.97 160.2 (1H, s, OH)  6 109.17 109.0,109.6  7 163.19 162.9  8 6.46 (1H, s)  93.15 6.49 (br. s), 93.0, 94.16.48 (br. s)  9 156.58, 156.37 156.4 10 103.43 103.2, 103.9  1′ 120.99121.2  2′ 7.90 (1H, d, 128.40 7.91 (d, 128.6 J = 8.78 Hz) J = 8.8)  3′6.90 (1H, d, 116.13 6.91 (d, 116.2 J = 8.78 Hz) J = 8.8)  4′ 161.39,161.25 161.4  5′ 6.90 (1H, d, 116.13 6.91 (d, 116.2 J = 8.78 Hz) J =8.8)  6′ 7.90 (1H, d, 128.40 7.91 (d, 128.6 J = 8.78 Hz) J = 8.8)  1″4.64 (1H,  71.69 4.66 (d,  71.0 J = 9.79 Hz) J = 10.2), 4.61 (d, J =9.5)  2″ 4.38 (1H, t, 75.81 76.20 4.37 (t, 74.4, 75.8 J = 9.54 Hz) J =9.0), 4.23 (1H, t, 4.18 (d, J = 9.54 Hz) J = 8.8)  3″ 3.34 80.11, 79.713.28-3.38 (m) 79.7, 79.9  4″ 3.16  70.01 3.05-3.15 (m) 70.5, 70.9  5″3.14  81.50 3.09-3.19 (m)  81.3  6″ 3.41, 3.67 61.26, 61.79 3.69 (d,61.3, 61.8 J = 11.1), 3.37-4.49 (m)  1″′ 5.04 (1H, d, 100.75, 100.405.00 (br. s), 100.2, 100.6 J = 30.37 Hz) 5.07 (br. s)  2″′ 3.60  70.693.60 (br. s),  70.5 3.31 (br. s)  3″′ 3.14  70.59 3.06-3.18 (m)  70.4 4″′ 2.91  71.69 2.91 (t,  71.5 (1H, t, J = 8.5), 9.16 Hz) 2.90 (t, J =8.5)  5″′ 2.31 (1H, m)  68.28 2.26-2.36 (m)  68.3  6″′ 0.61 (3H, d,17.82, 17.60 0.51 (d,  17.7 J = 5.77 Hz), J = 5.9), 0.53 (3H, d, 0.59(d, J = 7.03 Hz) J = 5.9)

Since the NMR chemical shifts were in agreement with reported values,Compound P9 was identified as isovitexin-2″-O-rhamnoside.

Compound P7

As shown in FIG. 4, when subject to ESI/MS (positive ion mode), compoundP7 displayed a molecular ion (MI) at m/z 595.4 [M+H]⁺ and a base peak(BP) at m/z 617.4 [M+Na]⁺. Its molecular formula was deduced asC₂₇H₃₀O₁₅. Fragmentations of molecular ion (FIG. 6) and base peak (FIG.5) produced abundant ions Yo⁺; 433.0 [(M+H)-146]⁺ and 455.3[(M+Na)-162]⁺. Such masses are attributed to loss of a neutral sugarmoiety (hexose) from glycosylated hydroxyl groups (Ferreres et al., 1161J Chromatogr A 214-23 (2007); Waridel et al., 926 J Chromatogr A. 29-41(2001)). Ions typical of C-hexosyl flavones (see FIGS. 5-6 and Table 3)were also observed: ^(0.3)X₀ ⁺: 365.3 [(M+Na)-162-90]⁺ and 343.0[(M+H)-162-90]⁺; and ^(0.2)X₀ ⁺: 335.3 [(M+Na)-162-120]⁺ and 313.1[(M+H)-162-120]⁺ (Ferreres et al., 1161 J Chromatogr A 214-23 (2007)).

TABLE 3 Experimental data No. ESI(+)-MS ESI(+)-MS/MS  1 617.4[M + Na]⁺617.3 [M + Na]⁺  2 599.4[(M + Na)-H₂0]⁺  3 497.3[(M + Na)-120]⁺  4455.2[(M + Na)-162 (Glu)]⁺→ Y_(o) ⁺  5 437.2[(M + Na)-Glu-H₂0]⁺  6419.2[(M + Na)-Glu-2 H₂0]⁺  7 365.2[(M + Na)- Glu-90]⁺ →^(0,3)χ₀ ⁺  8595.4 [M + H]⁺ 595.1 [M + H]⁺  9 433.0[(M + H)-162 (Glu)]⁺ → Y_(o) ⁺ 10415.0[(M + H)- Glu -H₂O]⁺ 11 367.0 [(M + H)- Glu -66]⁺→^(2,3)X⁺-2H₂O 12313.1 [(M + H)- Glu -120]⁺ →^(0,3)X₀ ⁺

Compound P7 was also characterized by ¹H NMR and ¹³C NMR. The ¹H NMRspectrum shows that a hexose is linked to position 6 (flavone) since H-8is displayed as a singlet at δ 6.47 ppm. See Table 4 (providing NMR datafor compound P7).

TABLE 4 Experimental Data ¹H δ (ppm) ¹³C δ (ppm) DMSO-d₆, DMSO-d₆, No.400 MHz 100 MHz  2 163.31  3 6.77 (1H, s) 102.72  4 181.74  5 13.68 (1OH, br s) 162.31 (HMBC) 13.55 (1 OH, br s)  6 108.05  7 163.23  8 6.47(1H, s) 93.98, 93.02  9 156.40 10 103.42 (HMBC)  1′ 121.16  2′ 7.94 (1H,d, 128.43 J = 8.8 Hz)  3′ 6.93 (1H, d, 116.0 J = 8.8 Hz)  4′ 161.19  5′6.93 (1H, d, 116.0 J = 8.8 Hz)  6′ 7.94 (1H, d, 128.43 J = 8.8 Hz)  1″4.65 (1H, d, 71.17 J = 9.8 Hz)  2″ 4.42 81.31  3″ 3.42 78.58  4″ 3.1570.72  5″ 3.15 81.62  6″ 3.68 (1H, d, 61.33 J = 10.5 Hz), 3.38  1″′ 4.11106.3  2″′ 2.94 74.44  3″′ 3.19 72.19  4″′ 3.19 73.36  5″′ 3.51 66.98 6″′ 3.12, 2.80 58.43

The HMBC spectra (not shown) of compound P7 indicate that compound P7has a hexose link through an O-glycosidic bond at position 2″. Thecompound P7 is identified as isovitexin-2″-O-glucoside.

Compound P8

As shown in FIG. 7, when subject to ESI/MS (positive ion mode), compoundP8 displayed a molecular ion (MI) at m/z 565.2 [M+H]⁺ and a base peak(BP) at m/z 587.3 [M+Na]⁺. Its molecular formula was deduced asC₂₆H₂₈O₁₄. Fragmentations of molecular ion (FIG. 9) produced an abundantion Yo⁺; 433.0=[(M+H)-132]⁺, which can be attributed to a loss of aneutral sugar moiety (pentose) from glycosylation on hydroxyl groups(Ferreres et al., 1161 J Chromatogr A 214-23 (2007); Waridel et al., 926J Chromatogr A. 29-41 (2001)). The fragmentation pattern of the basepeak is shown in FIG. 8. Ions typical of C-hexosyl flavones (see FIGS.8-9 and Table 5) were also observed: ^(0.3)X₀ ⁺: 365.3 [(M+Na)-132-90]⁺,^(0.2)X₀ ⁺: 335.3 [(M+Na)-132-120]⁺ and 313.1 [(M+H)-132-120]⁺ (Ferrereset al., 1161 J Chromatogr A 214-23 (2007).

TABLE 5 Experimental data No. ESI(+)-MS ESI(+)-MS/MS  1 587.4[M + Na]⁺587.3[M + Na]⁺  2 569.4[(M + Na)-H₂0]⁺  3 467.3[(M + Na)-120]⁺  4455.3[(M + Na)-132 (Xyl)]⁺ →Y_(o) ⁺  5 437.3[(M + Na)-Xyl-H₂0]⁺  6 365.3[(M + Na)-Xyl-90]⁺ → ^(0,3)X⁺  7 335.3 [(M + Na)-Xyl-120]⁺ → ^(0,2)X⁺  8565.3 [M + H]⁺ 565.3[M + H]⁺  9 547.3[(M + H)- H₂0]⁺ 10 433.2[(M +H)-Xyl]⁺ →Y_(o) ⁺ 11 415.2[(M + H)-Xyl-H₂0]⁺ 12 397.2[(M + H)-Xyl-2H₂0]⁺13 367.2[(M + H)-Xyl-66]⁺ →^(2,3)X⁺ -2H₂O 14 337.2[(M + H)-Xyl-96]⁺→^(0,4)X⁺-2H₂O 15 313.2[(M + H)-Xyl-120]⁺ → ^(0,2)X⁺ 16 283.2[(M +H)-Xyl-150]⁺ → ^(0,1)X⁺ 17 271.3[(M + H)-Xyl-Glu]⁺

Additionally, as shown in FIG. 9, compound P8 displayed fragment ionsassociated to a pentose moiety neutral loss and a hexose cleavage: 415.1[(M+H)-132-H₂O]⁺, 397.1 [(M+H)-132-2H₂O]⁺, 367.1[(M+H)-132-66]⁺→^(2,3)X⁺→^(2,3)H₂O, 337.1 [(M+H)-132-96]⁺→^(0,4)X⁺-2H₂Oy 283.1 [(M+H)-132-150]⁺→^(0,1)X⁺.

Compound P8 was also characterized by ¹H NMR and ¹³C NMR. Theexperimental results are shown in Table 6 alongside published date forisovitexin-2″-xyloside (Zielinska-Pisklak et al., 102 J Pharm BiomedAnal 54-63 (2015)).

TABLE 6 Isovitexin 2″-xyloside (Zielińska-Pisklak et al., 102 J PharmBiomed Experimental data Anal 54-63 (2015)) ¹H δ (ppm) ¹³C δ ppm) ¹H δ(ppm) ¹³C δ (ppm) DMSO-d₆, DMSO-d₆, DMSO-d₆, DMSO-d₆, No. 400 MHz 100MHz 300 MHz, 298K 750 MHz, 298K  2 163.36 163.44  3 6.74 (1H, s) 102.736.78 (1H, s) 102.69  4 181.90 181.88  5 13.66, 13.57 162.19 13.68 160.48(OH) (1H, s, OH)  6 108.17 107.97  7 162.86 (HMBC H-8) 161.74  8 6.45(1H, s) 93.82, 93.12 6.54 (1H, s) 93.82  9 156.45 156.34 10 103.20 10.95103.17 (1H, s, OH)  1′ 121.11 121.04  2′ 7.90 (1H, d, 128.45 7.93 (1H,d, 128.42 J = 8.8 Hz) J = 8.8 Hz)  3′ 6.91 (1H, d, 116.02 6.94 (1H, d,115.99 J = 8.8 Hz) J = 8.8 Hz)  4′ 161.22 10.44 161.2 (1H, s, OH)  5′6.91 (1H, d, 116.02 6.94 (1H, d, 115.99 J = 8.8 Hz) J = 8.8 Hz)  6′ 7.90(1H, d, 128.45 7.93 (1H, d, 128.42 J = 8.8 Hz) J = 8.8 Hz)  1″ 4.65 (1H,71.42 4.66 (1H, d, 71.23 d, 9.8 Hz) J = 9.8 Hz)  2″ 4.38 80.22 4.42-3.17(6H, 81.58 overlapped)  3″ 3.42 78.69 78.33  4″ 3.17 70.35 70.35  5″3.17 81.62 80.96  6″ 3.68 (1H, d, 61.52 61.37 11.54 Hz), 3.39  1″′ 4.23(1H, d, 105.23 4.13 (1H, d, 105.97 6.02 Hz) J = 7.2 Hz)  2′″ 3.26 72.39(¹³C) 3.17-2.57 (5H, 74.18 overlapped)  3″′ 3.30 71.23 76.20  4″′ 3.4266.58 69.30 (66.89 ¹³C)  5″′ 3.02, 2.88 64.48 65.67

As the NMR chemical shifts were in agreement with reported values (seeTable 6), compound P8 was identified as isovitexin-2″-O-xyloside(Zielinska-Pisklak et al., 102 J Pharm Biomed Anal 54-63 (2015).

Compound P6

As shown in FIG. 10 and Table 7, when subjected to ESI/MS (positive ionmode), compound P6 displayed a molecular ion (MI) at m/z 565.2 [M+H]⁺and a base peak (BP) at m/z 587.3 [M+Na]⁺ that closely correspond withvalues obtained for compound P8. The molecular formula of compound P6was deduced to be C₂₆H₂₈O₁₄. Fragmentations of molecular ion (FIG. 12)produced an abundant ion Yo⁺; 433.0=[(M+H)-132]⁺, attributed to loss ofa neutral sugar moiety (pentose) from glycosylated on hydroxyl groups(Ferreres et al., 1161 J Chromatogr A 214-23 (2007); Waridel et al., 926J Chromatogr A. 29-41 (2001)). Ions typical of C-hexosyl flavones (seeFIGS. 11 and 12) were also observed: ^(0.3)X₀ ⁺: 365.3 [(M+Na)-132-90]⁺,^(0.2)X₀ ⁺: 335.3 [(M+Na)-132-120]⁺ and 313.1 [(M+H)-132-120]⁺ (Ferrereset al., 1161 J Chromatogr A 214-23 (2007)).

TABLE 7 Experimental data No. ESI(+)-MS ESI(+)-MS/MS  1 587.3[M + Na]⁺587.2[M + Na]⁺  2 569.2[(M + Na)-H₂0]⁺  3 467.1[(M + Na)-120]⁺  4455.1[(M + Na)-132 (Xyl)]⁺  5 437.2[(M + Na)-Xyl-H₂0]⁺  6 365.2 [(M +Na)-Xyl-90]⁺  7 335.2 [(M + Na)-Xyl-120]⁺  8 565.2 [M + H]⁺ 565.2[M +H]⁺  9 547.2[(M + H)- H₂0]⁺ 10 445.1[(M + H)-120]⁺ 11 433.1[(M +H)-Xyl]⁺ 12 415.1[(M + H)-Xyl-H₂0]⁺ 13 397.1[(M + H)-Xyl-2H₂0]⁺ 14367.1[(M + H)-Xyl-66]⁺→^(2,3)X⁺-2H₂O 15 337.1[(M + H)-Xyl-96]⁺→^(0,4)χ⁺-2H₂O 16 313.1[(M + H)-Xyl-120]⁺ 17 283.1[(M + H)-Xyl-150]⁺→^(0,1)χ⁺

Additionally, as shown in FIG. 12, compound P6 displayed fragment ionsassociated to a pentose moiety neutral loss and a hexose cleavage: 415.1[(M+H)-132-H₂O]⁺, 397.1 [(M+H)-132-2H₂O]⁺, 367.1[(M+H)-132-66]⁺→^(2,3)X⁺-2H₂O, 337.1 [(M+H)-132-96]⁺→^(0,4)X⁺-2H₂O and283.1 [(M+H)-132-150]⁺→^(0,1)X⁺.

Compound P6 was also characterized by both ¹H NMR. The experimentalresults are shown in FIG. 13 and Table 8.

TABLE 8 Experimental data ¹H δ (ppm) DMSO-d₆, 400 MHz No. Compound P6Compound P8 3 6.81 (1H, s) 6.74 (1H, s) 5 13.54 (1H, br s, 13.66, 13.57OH) (OH) 8 6.37 (1H, s) 6.45 (1H, s) 2′ 7.77 (1H, d, 7.90 (1H, d, J =8.7 Hz) J = 8.8 Hz) 3′ 6.85 (1H, d, 6.91 (1H, d, J = 8.7 Hz) J = 8.8 Hz)5′ 6.85 (1H, d, 6.91 (1H, d, J = 8.7 Hz) J = 8.8 Hz) 6′ 7.77 (1H, d,7.90 (1H, d, J = 8.7 HZ) J = 8.8 Hz) 1″ 4.66 4.65 (1H, d, 9.8 Hz) 2″4.39 4.38 3″ 3.44 3.42 4″ 3.17 3.17 5″ 3.20 3.17 6″ * 3.68 (1H, d, 11.54Hz), 3.39 1″′ 4.16 4.23 (1H, d, 6.02 Hz) 2″′ * 3.26 3″′ * 3.30 4″′ *3.42 5″′ * 3.02, 2.88 Note: * No identified chemical shifts.

The ¹H NMR spectrum displayed similar chemical shifts to compound P8.These results suggest that compound P6 is an isomer of compound P8.Accordingly, compound P6 is identified as isovitexin-O-xyloside.

Compound P4

As shown in both FIG. 14 and Table 9, when subjected to ESI/MS (positiveion mode) compound P4 displayed a molecular ion (MI) at m/z 581.2 [M+H]⁺and a base peak (BP) at m/z 603.2 [M+Na]⁺. Its molecular formula wasdeduced as C₂₆H₂₈O₁₅. Fragmentation of molecular ion (FIG. 15) producedan abundant ion Yo⁺; 449.2=[(M+H)−132]⁺, which was attributed to loss ofa neutral sugar moiety (pentose) from glycosylated hydroxyl groups(Ferreres et al., 1161 J Chromatogr A 214-23 (2007); Waridel et al., 926J Chromatogr A. 29-41 (2001)). As shown in Table 9 and FIGS. 15 and 16,ions typical of C-hexosyl flavones were also observed: E₁: 431.1[(M+H)-Xyl-H₂O]⁺, E₂: 413.1 [(M+H)-Xyl-2H₂O]⁺, E₃: 395.1[(M+H)-Xyl-3H₂O]⁺, ^(2,3)X+-2H₂O: 383.1 [(M+H)-Xyl-66]⁺, ^(0,4)X-2H₂O:353.1 [(M+H)-Xyl-96]⁺ and ^(0.2)X⁺: 329.1 [(M+H)-Xyl-120]⁺ (Ferreres etal., 1161 J Chromatogr A 214-23 (2007).

TABLE 9 Experimental data No. MALDI(+)-MS MALDI(+)-MS/MS 1 603.2 [M +Na]⁺ 581.2 [M + H]⁺: 2 581.2 [M + H]⁺ a. 563.2[(M + H)-H₂O]⁺ 3449.1[(M + H)-132 (Xyl)]⁺→ Y_(o) ⁺ b. 461.2[(M + H)-120]⁺ →^(0,2)X⁺ 4431.1 [(M + H)- Xyl -H₂O]⁺ c. 449.2[(M + H)-132 (Xyl)]⁺→ Y_(o) ⁺ 5 413.1[(M + H)- Xyl -2H₂O]⁺ d. 431.2 [(M + H)- Xyl -H₂O]⁺ 6 395.1 [(M + H)-Xyl-3H₂O]⁺ e. 353.1 [(M + H)-Xyl-96]⁺→ (^(0,4)X- 2H₂O)⁺ 7 383.1[(M +H)-Xyl-66]⁺→ (^(2,3)X⁺- f. 329.1[(M + H)-Xyl-120]⁺ → ^(0,2)X⁺ 2H₂O) 8353.1 [(M + H)-Xyl-96]⁺→ (^(0,4)X- g. 287.1[(M + H)-Xyl-Glu]⁺ 2H₂O)⁺ 9329.1 [(M + H)-Xyl-120]⁺ → ^(0,2)X⁺ 329.1 [(M + H)-Xyl-120]⁺→^(0,2)X⁺ a.329.1: ^(0,2)X⁺ b. 300.2: ^(0,2)X⁺ - CHO c. 283.1: ^(0,2)X⁺ - CH₂O₂ d.195.1: ^(1,3)A⁺ e. 177.1: ^(1,3)A⁺ - H₂O f. 137.1: ^(0,2)B⁺

Compound P4 was also characterized by ¹H NMR. The experimental resultsare shown in Table 10 alongside published data forisoorientin-2″-O-xyloside (Matsuzaki et al., 44 Japanese Soc Pharmacogn251-253 (1990)).

TABLE 10 Published data Isoorientin-2″-O-xyloside Experimental data(Matsuzaki et al., 44 ¹H δ (ppm) Japanese Soc Pharmacogn DMSO-d 251-253(1990)) ¹H δ (ppm) No. 400 MHz DMSO-d 400 MHz 3 6.65 (1H, s) 6.68 (1H,s) 5 13.55 (1H, br. s, 13.70 (1H, br. s, OH) OH) 8 6.43 (1H, s) 6.46(1H, s) 7 — — 2′ 7.39 (1H, d, 7.40-7.43 J = 2.5 Hz) (1H, m) 3′ — — 4′ —— 5′ 6.87 (1H, d, — J = 2.1 Hz) 6′ 7.42 (1H, d, 7.40-7.43 J = 2.1 Hz)(1H, m) 1″ 4.63 (1H, d, 4.64 (1H, d, J = 9.79 Hz) J = 9.6 Hz) 2″ * —3″ * — 4″ * — 5″ * — 6″ * — 1″′  4.11 (1H, d, , 4.12 (1H, d, J = 6.02Hz) J = 6.7 Hz) 2″′ * — 3″′ * — 4″′ * — 5″′ * — Note: * No identifiedchemical shifts. —Not reported

The ¹H NMR spectrum shows that a hexose is attached to position 6 of theflavone since H-8 appeared as a singlet at δ 6.43 ppm (Matsuzaki et al.,44 Japanese Soc Pharmacogn 251-253 (1990)). In addition, 5-OH isdisplayed as a broad singlet at δ 13.49 ppm (Doyama et al., 96 JEthnopharmacol 371-4 (2005); Wen et al., 2 Asian J Tradit Med. 149-53(2007)). The compound P4 is identified as isoorientin-2″-O-xyloside.

Compound P2

As shown in FIG. 17, when subjected to ESI/MS (positive ion mode),compound P2 displayed a base peak (BP) at m/z 377.2 [M+Na]⁺. Itsmolecular formula was deducted as C₁₆H₁₈O₉. The compound P2 isidentified as chlorogenic acid by direct comparison (retention time)with an analytical standard (FIG. 18).

Perturbation of G-Protein Coupled Receptors

Angiotensin II, receptor type 1 (“AT₁”), angiotensin II, receptor type 2(“AT₂”), and endothelin receptor type B (“ET_(B)”) are G-protein coupledreceptors that play a role in blood pressure regulation and vascularremodeling. Activation of the AT₁ receptor can lead to vasoconstriction,aldosterone synthesis and secretion, increased vasopressin secretion,cardiac hypertrophy, augmentation of peripheral noradrenergic activity,vascular smooth muscle cells proliferation, decreased renal blood flow,renal renin inhibition, renal tubular sodium reuptake, modulation ofcentral sympathetic nervous system activity, cardiac contractility,central osmocontrol, and extracellular matrix formation (Catt et al., JCardiovasc Pharmacol. 6 Suppl 4:S575-86 (1984). Activation of the AT₂receptor can induce vasodilation in multiple vascular beds, enhancenatriuresis, prevent vascular remodeling by decreasing collagendeposition, and attenuate arterial stiffening. Activation of the ET_(B)receptor can lead to vasodilation and clearance of endothelin 1 from thesystemic blood.

To investigate the vasculoprotective, neuroprotective, and/orantihypertensive properties of Cecropia extracts, Chinese hamster ovarycells that had been stably transfected with aequorin (CHO-AEQ cells)were transiently transfected with various G-protein coupled receptors.

More particularly, CHO-AEQ cells were cultured in Dulbecco's ModifiedEagle's Medium (DMEM) containing 10% fetal bovine serum as previouslydescribed (Vanderheyden et al., 126 Br J Pharmacol 1057-1065 (1999)). At75% confluency, these cells were transiently transfected with pcDNA3.1+plasmid DNA into which had been inserted the coding region for humanAT₁, AT₂, or ET_(B) receptor. The transient transfection was performedwith Fugene HD® following the manufacturers' instructions (2 μg DNA to 7μl Fugene HD® in Opti-MEM® medium. After transfection, the cells werecultured for an additional day, after which they were harvested andloaded with coelenterazine-h.

Intracellular calcium can be detected based on the interaction ofcalcium ions with the calcium binding bioluminescent complex aequorin.Upon calcium binding, aequorin oxidizes coelenterazine-h intocoelenteramide with production of CO₂ and emission of light (466 nm) (LePoul et al., 7 J Biomol Screen 57-65 (2002). The CHO-AEQ cells that hadbeen transiently transfected with human AT₁, human AT₂ or human ET_(B)receptors were used in a bioluminescence assay to evaluate whether—andto what extent—material isolated from Cecropia obtusifolia could perturbG-protein coupled receptor(s).

Confluent CHO-AEQ cells that were grown and transfected in 75 cm²culture flasks were harvested by a brief treatment with a trypsin/EDTAsolution, centrifuged, and resuspended in DMEM-F12 cell culture mediumcontaining 0.1% BSA and denoted as assay buffer, at a cell density of2.5×10⁶ cells/ml. Subsequently, coelenterazine-h was added to the cellsuspension to a final concentration of 5 μM. Cells were then incubatedbetween 14 h and 18 h at room temperature in the dark under gentleshaking. After this loading step, 15 ml of assay buffer was added to thecells and then centrifuged for 7 min at 1100 rpm in a swinging bucketcentrifuge. The resulting cell pellets were gently resuspended in assaybuffer containing 1 μM coelenterazine-h at a cell density of 5×10⁵cells/ml. After a further incubation of 1.5 h at room temperature, thecell suspension was distributed at 100 μl/well in white Cellstar 96-wellplates.

To assess the activity of the compounds derived from Cecropiaobtusifolia extracts, lyophilized crude extract (and compounds that hadbeen identified in the extract) were dissolved in DMSO at 1 mg/ml, andthen further diluted in assay buffer to 100 μg/ml (final concentration)(Caballero-George et al., 4 Curr. Trends Biotechnol Pharm 881-899(2010)). A 50 aliquot was then added to each well containing thetransiently transfected cells. For comparison, control peptides werediluted in assay buffer to a final concentration of 0.1 μM angiotensinII or 10 nM endothelin-1. These control peptides are known to cause atransient rise in cytosolic calcium. The agonist solutions were loadedin the cell injector of a Victor spectrophotometer, and a 96-well platewas inserted.

Measurements were initiated by injecting 50 μl solution/well, and thebioluminescence was recorded every 200 ms for 120 cycles. To account forslight variations in cell number, a final concentration of 10 μM ATP wasadded by a second injector to the cells (50 μl/well) and the resultingresponse was measured.

The effects were quantified by calculation of the area under the curvesby integration of the obtained transient responses using GraphPad Prism5™. The activation of the AT₁, AT₂, or ET_(B) receptors was expressed asa fractional response. This is obtained by dividing the agonist responseby the sum of the agonist and ATP response for each well (i.e., agonistresponse/(ATP+agonist response)). The fractional response is used tonormalize the agonist response in each well for the amount of livingcells in the well (that is proportional to the ATP response) (Nikolaou35 al., 702 Eur J Pharmacol 93-102 (2013)).

The effect of the evaluated extracts was calculated as percentinhibition of the control responses and is given as the average±standarderror of three determinations. In order to validate the three receptorassays, the agonist responses were measured after pre-incubation with0.1 μM BQ788 or 100 μM losartan, which are selective antagonists for theAT₂, ET_(B), and AT₁ receptors, respectively.

The results are shown in FIGS. 17-19. In each figure, the Y-axisrepresents the amount of cytoplasmic calcium expressed as a fractionfrom the total intracellular content.

More particularly, FIG. 19 shows cytoplasmic calcium release afterbinding of the compounds or extracts to AT₂ receptors expressed onCHO-AEQ cells. Extract of the Cecropia genus containing compounds P2,P4, P6, P7, P8 and P9 showed a higher degree of activation of the AT₂receptor than the AT₂ endogenous peptide angiotensin II. Compounds P2,P4, P6, P7, P8 and P9, individually tested, showed approximately half ofthe activity of the extract suggesting synergism of the individualcomponents of Cecropia genus active extract on the activation of the AT₂receptor.

FIG. 20 shows cytoplasmic calcium release after binding of the compoundsor extracts to the ET_(B) receptors expressed on CHO-AEQ cells. Fraction3-4—which is composed mainly of compounds P9 and P8—induced a highercytoplasmic calcium release than 100 nM of the endogenous agonistendothelin 1 (ET-1). In this experiment, compound P6, which is not theprimary component of the extract, is the most active sample tested,followed by P4, P7, P8, P9 and P2.

FIG. 21 shows that Fraction 3-4 inhibits the agonistic effect of 100 nMangiotensin II on the AT₁ receptor with the same intensity as theselective AT₁ receptor antagonist losartan. Flavonoids P2, P4, P7, P8and P9 from this fraction inhibited angiotensin II effect on the AT₁receptor with different intensities. Their effect is less than half ofthe effect of Fraction 3-4 when tested individually, suggesting asynergistic effect of these components.

Any methods disclosed herein include one or more steps or actions forperforming the described method. The method steps and/or actions may beinterchanged with one another. In other words, unless a specific orderof steps or actions is required for proper operation of the embodiment,the order and/or use of specific steps and/or actions may be modified.Moreover, sub-routines or only a portion of a method described hereinmay be a separate method within the scope of this disclosure. Statedotherwise, some methods may include only a portion of the stepsdescribed in a more detailed method.

Reference throughout this specification to “an embodiment” or “theembodiment” means that a particular feature, structure, orcharacteristic described in connection with that embodiment is includedin at least one embodiment. Thus, the quoted phrases, or variationsthereof, as recited throughout this specification are not necessarilyall referring to the same embodiment.

Similarly, it should be appreciated by one of skill in the art with thebenefit of this disclosure that in the above description of embodiments,various features are sometimes grouped together in a single embodiment,figure, or description thereof for the purpose of streamlining thedisclosure. This method of disclosure, however, is not to be interpretedas reflecting an intention that any claim requires more features thanthose expressly recited in that claim. Rather, as the following claimsreflect, inventive aspects lie in a combination of fewer than allfeatures of any single foregoing disclosed embodiment. Thus, the claimsfollowing this Detailed Description are hereby expressly incorporatedinto this Detailed Description, with each claim standing on its own as aseparate embodiment. This disclosure includes all permutations of theindependent claims with their dependent claims.

Recitation in the claims of the term “first” with respect to a featureor element does not necessarily imply the existence of a second oradditional such feature or element. It will be apparent to those havingskill in the art that changes may be made to the details of theabove-described embodiments without departing from the underlyingprinciples of the present disclosure.

1. A method for producing a formulation for administration to amammalian subject, the method comprising: mixing plant material with analcohol, wherein the plant material is from one or more plants belongingto the Cecropia genus; extracting a portion of the plant material intothe alcohol; isolating the extracted portion of plant material from aremainder of the plant material; and removing at least a portion of theisolated extracted portion of the plant material to obtain a firstcomposition, the extracted portion having two or more flavonoids, thetwo or more flavonoids in the formulation being selected only from thegroup consisting of isovitexin-2″-O-rhamnoside,isovitexin-2″-O-glucoside, isovitexin-2″-O-xyloside,isovitexin-O-xyloside, isoorientin-2″-O-xyloside, and mixtures thereof.2. The method of claim 1, wherein the two or more flavonoidscollectively (1) antagonize the angiotensin II receptor, type 1, (2)agonize the angiotensin II receptor, type 2, and (3) agonize theendothelin receptor type B.
 3. The method of claim 1, further comprisingcombining the first composition with a second composition to form theformulation for administration to the mammalian subject.
 4. The methodof claim 1, wherein the alcohol has four or fewer carbon atoms.
 5. Themethod of claim 1, wherein the alcohol is methanol.
 6. The method ofclaim 1, wherein the extracted portion is further washed with a solventcomprising dichloromethane.
 7. The method of claim 1, wherein theextracted portion is further washed with a solvent comprising hexane. 8.The method of claim 1, wherein the extracted portion is further washedwith a solvent comprising hexane.
 9. The method of claim 1, wherein theisolating the extracted portion of plant material is carried out viachromatography.
 10. The method of claim 1, wherein the plant material ismacerated prior to extracting a portion of the plant material into theone or more organic solvents.
 11. The method of claim 1, wherein the twoor more flavonoids are between 0.3% and 15% of the formulation byweight.
 12. The method of claim 1, wherein the plant material isexclusively from aerial parts of the plant.
 13. The method of claim 1,wherein the formulation lowers blood pressure when administered to themammalian subject.
 14. The method of claim 1, one or more plants belongto the species Cecropia obtusifolia.